Sakurai et al. BMC Medical Genetics(2020) 21:201
https://doi.org/10.1186/s12881-020-01140-9

RESEARCH ARTICLE

Open AccessInfluence of MIF polymorphisms on CpG
island hyper-methylation of CDKN2A in thepatients with ulcerative colitis
Naoko Sakurai1, Tomoyuki Shibata2, Masakatsu Nakamura1, Hikaru Takano1, Tasuku Hayashi1, Masafumi Ota1,
Tomoe Nomura-Horita1, Ranji Hayashi1, Takeo Shimasaki1, Toshimi Ostuka1, Tomomitsu Tahara3 andTomiyasu Arisawa1*

Abstract
Background: CDKN2A hypermethylation is among the major events associated with carcinogenesis and is also
observed in non-neoplastic colonic mucosa in patients with ulcerative colitis (UC). Macrophage migration inhibitory
factor (MIF) plays a crucial role in promoting gastrointestinal inflammation characteristic of UC. The aim of thisstudy is to explore associations between CDKN2A methylation status and MIF polymorphisms (rs755622 andrs5844572).
Methods: One hundred and fifty-nine patients diagnosed with UC were enrolled in this study. The methylation
status of p14ARF and p16INK4a was determined by MSP; MIF genotypes were identified by PCR-SSCP.
Results: We found no differences with respect to mean age, gender, clinical type (chronic continuous or relapse/
remitting), or extent of disease among the patients with methylated and unmethylated p14ARF or p16INK4a. Carrying
the rs755622 C allele indicated a significantly higher risk for p14ARF methylation (odds ratio (OR), 2.16; 95%
confidence interval (CI), 1.08–4.32; p = 0.030); similarly, carrying the rs5844572 7-repeat allele indicated a significantly
higher risk for p16INK4a methylation (OR, 2.57; 95% CI, 1.26–5.24; p = 0.0094) after an adjusted regression analysis. The
carriers of the rs755662 C allele or the rs5844572 7-repeat allele were both at a significantly higher risk formethylation of both p14ARF and p16INK4a when compared to the cohort in which neither of the genes were
methylated (OR, 2.70; 95% CI, 1.22–6.01; p = 0.015 and OR, 2.87; 95% CI, 1.25–6.62; p = 0.013, respectively).
Additionally, carrying rs755622 C allele was significantly associated with CIHM in chronic continuous of clinical type
and total colitis (OR, 25.9; 95% CI, 2.55–262.6; p = 0.0059 and OR, 4.38; 95% CI, 1.12–17.2; p = 0.034, respectively), andcarrying 7-repeat allele of rs5844572 was significantly associated in chronic continuous type (OR, 14.5; 95%CI, 1.46–
144.3; p = 0.022).
Conclusions: Taken together, our findings suggest that MIF genotypes associated with inflammation may also be
involved in promoting carcinogenesis via CDKN2A hypermethylation in patients diagnosed with UC.
Keywords: Ulcerative colitis, CDKN2A, CpG hypermethylation, Macrophage migration inhibitory factor, Genetic
polymorphism* Correspondence: tarisawa@kanazawa-med.ac.jp
1
Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku,
Uchinada-machi, Ishikawa 920-0293, Japan
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Sakurai et al. BMC Medical Genetics(2020) 21:201

BackgroundUlcerative colitis (UC) is nonspecific inflammation of
the large intestine with unknown etiology and its inflammation may involve the colonic mucosa spanning fromthe rectum to the cecum [1]. UC patients have a chronicor remission/relapsing course and many inflammationor immune- related factors attribute to the severity of
inflammation. Macrophage migration inhibitory factor(MIF) was initially identified as a factor released by T
cells that inhibits the random migration of macrophages[2]. Subsequent studies revealed that MIF is a proinflammatory factor, which has important roles invarious chronic inflammatory diseases and immune
disorders, including UC [3, 4]. In particular, Ishiguro
et al. reported that MIF contributes to steroid resistanceof refractory UC via activator protein (AP)-1 signaling[5]. Two distinct polymorphisms were identified in MIF:
rs755622 (− 173 G > C) and rs5844572 (− 794 CATT tandem repeat), that were found to be in linkage disequilibrium [6]. Our previous study revealed that these genetic
polymorphisms had little influence on the susceptibility
to UC [7]; however, a recent meta-analysis based on recessive and co-dominant genetic models identified a significant relationship linking the rs755622 polymorphismand susceptibility to disease [8, 9]. As such, the MIF
genotype seems to influence the development and progression of UC.
Recent advances with respect to our understanding thepathogenesis of UC together with the development of
new therapeutic agents have introduced the possibilityof disease control in many cases of UC [10]. However, as
the incidence of colitis-associated-cancer (CAC) increases among patients with UC in proportion to theduration of the disease [11, 12], prevention of carcinogenesis and identification of high-risk groups are currently essential clinical issues. Generally, important risk
factors for development of CAC are the existence of extensive colonic lesions [11], longer duration of disease[12], positive family history of colorectal cancers [13, 14]
and the presence of histologically-active inflammation[15]. However, the risk factors underlying UC-associatedcarcinogenesis require further and ongoing clarification.
CpG island hypermethylation (CIHM) is a critical mechanism that promotes gene inactivation and is commonly
observed in association with numerous human cancers[16]. Additionally, CIHM of several specific genes, aphenomenon known as age-related methylation, was also
detected in non-neoplastic tissues [17]; this type ofmethylation has been related to precancerous states [18].
CIHM has been reported within non-neoplastic colonicmucosal tissues of patients diagnosed with UC; likewise,
chronic inflammation has been shown to promote agerelated methylation [19]. Our previously study also revealed aberrant methylation of the tumor suppressors

Page 2 of 9

p14ARF and p16INK4a, both encoded by Cyclin Dependent
Kinase Inhibitor 2A (CDKN2A) locus, in the nonneoplastic colonic mucosal tissues of patients with UC [20].
As such, we considered the possibility of identifying
patients at high risk for the development of CAC byexamining the impact of specific genotypes on CIHM ofthe genes associated with precancerous states. In thecurrent study, we explored the relationship between
polymorphisms of MIF, a gene encoding a proinflammatory mediator associated with UC, and CIHM
of p14ARF and p16INK4a. Our goal was to determine
whether the MIF gene polymorphisms have any implications for the assessment of UC patients at high risk for
carcinogenesis.

Methods
Patients and samples

One hundred and fifty-nine patients with UC were enrolled in this study. All patients were treated at theEndoscopic Center of Fujita Health University Hospital,
registered from January 2006 to December 2012. UC
was diagnosed according to the standard criteria such as
clinical, endoscopic, and histological features [21]. Whencolonoscopy was performed, the biopsy specimens of inflammatory mucosa were obtained from the rectum of
all patients and reserved in − 80 °C. All patients were inendoscopic remission clinically but mild or moderate inflammation without evidence of dysplasia or neoplasia
was shown by histopathological examinations showed in
all cases. Genomic DNA was isolated using the FlexiGene DNA Kit (QIAGEN GmbH, Hilden, Germany)from peripheral blood obtained at the same time ascolonoscopy. The protocol for the present study wasapproved by the Ethics Committee of Fujita Health University (HM18–094), and written consent was obtainedin all cases.
Classifications

The enrolled patients were classified into two groups, including chronic continuous and relapse/remitting phenotypes, according to their previous clinical course [22].
Patients were also classified by endoscopic features as
total or subtotal (distal or left side) colitis according to
the location and extent of the inflammatory lesions.
Detection of DNA methylation of p14ARF and p16INK4a by
methylation-specific PCR method (MSP)

CIHM of p14ARF and p16INK4a was assessed according to
the method previously described [23]. We treated genomic
DNA, which extracted from rectal biopsy specimens using
proteinase K, with sodium bisulfite using the BislFast DNA
Modification Kit for methylated DNA Detection (Toyobo,
Co., Ltd., Osaka, Japan). The primer sets used at MSP wereshown in Table 1. We determined the annealing

Sakurai et al. BMC Medical Genetics(2020) 21:201

Table 1 Primer sets used in this study
Primer sets for MSP
p14-UM_F

5′-gagtttggttttggaggtgg-3’

p14-UM_R

5′-aaccacaacaacaaacacccct-3’

p14-M_F5′-tgagtttggttttggaggtgg-3’

p14-M_R

5′-aaaaccacaacgacgaacg-3’

p16-UM_F

5′-ttattagagggtggggtggattgt-3’

p16-UM_R

5′-caaccccaaaccacaaccataa-3’

p16-M_F5′-ttattagagggtggggcggatcgc-3’

p16-M_R

5′-accccgaaccgcgaccgtaa-3’

Primer sets for MIF polymorphism detection
rs755622_F

5′-tctagccgccaagtggagaaca-3’

rs755622_R

5′-actgtggtcccgccttttgtga-3’

rs5844572_F

5′-tgatccagttgctgccttgtc-3’

rs5844572_R

5′-tccactaatggtaaactcggggac-3’

MSP Methylation specific RCR, UM Unmethylated, M Methylated, F Forward,
R Reverse

temperature and times using DNA from peripheral bloodof a young individual (as an unmethylated control) and its
DNA treated with SssI methylase (methylated control; New
England BioLabs Inc., Beverly, MA, USA). Using EX Taq
HS (Takara Bio, Shiga, Japan), the PCR was performed withthe addition of 0.1 μg of bisulfite-modified DNA in 20 μL of
a buffer. The PCR condition were an initial denaturing stepof 5 min at 95 °C, followed by 33 cycles of 30 s denaturing
at 95 °C, 1 min annealing at 64–68 °C according to primers
used, and 1 min extension at 72 °C, and a final 5 min extension step at 72 °C. To detect the band of MSP sample, we
performed electrophoresis of PCR products in 3.0% agarosegels stained with ethidium bromide. Then, fluorescence intensity of UV illumination was measured by a digital densitometer. The methylation ratio was calculated as the ratio
of intensities of the methylated band to methylated plus
unmethylated bands, and a ratio more than 50% was judged
as significantly methylated.
Genotyping of MIF polymorphisms

The genotype of MIF polymorphisms was determined by
the polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) method as described
previously [7]. The primer sets used were shown in
Table 1. The PCR was performed using EX Taq HS(Takara Bio, Shiga, Japan), adding 0.1 μg of genomic
DNA extracted from peripheral blood to 20 μL of a buffer, denaturing at 95 °C for 3 min, followed by 35 cyclesof 15 s at 96 °C, 40s at 60 °C for rs755622 or 62 °C for
rs5844572, and 30 s at 72 °C, and 5 min final extension
at 72 °C. Then, 2 μL of the PCR product was treated in
10 μL of formamide for 5 min at 90 °C and denatured toa single strand. SSCP was performed in Gene Phor DNA
separation system using the Gene Gel Excel 12.5/24 kit

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(GE Health Care Bio-Sciences AB, Stockholm, Sweden)
at a constant temperature of 6 °C, and the denatured
bands were detected using the DNA silver staining kit(GE Health Care Bio-Sciences AB).
Statistical analysis

The Hardy–Weinberg equilibrium (HWE) was assessedby χ2 statistics. Mean age was expressed as mean ± SD
and analyzed by Student’s t-test. The ratio of sex and
CIHM frequencies was compared by Fisher’s exact test.
Allele counts and genotype distribution were also
compared between two groups by Fisher’s exact test.
The odds ratio (OR) and 95% confidence intervals (CI)for the strength of genotype involvement in CIHM were
calculated using a logistic regression analysis adjusted
for age, sex, clinical type and disease extension. A probability value of less than 0.05 was considered statisticallysignificant in all analyses. Stata software (version 13;
StataCorp LP, College Station, TX, USA) was used forstatistical processing.

Results
Demographic characteristics, allelic frequencies, and
CDKN2A methylation status

The characteristics and allele frequencies observed
among the UC patients enrolled in this study are shownin Table 2. The allele distribution of MIF (rs755622) met
the criteria for HWE (p = 1.00). We found no differenceswith respect to mean age, gender, clinical type, or extent
of disease among those with methylated and unmethylated p14ARF or p16INK4a. The minor allele frequency of
rs755622 was somewhat higher in the group with p14ARF
methylation; of note, the frequency of the C allele carrier
was significantly higher (p = 0.01). By contrast, no significant differences in the minor allele frequencies associated with rs755622 were observed when comparing thep16INK4a methylated and unmethylated groups. Similarly,
the 7-repeat allele frequency of rs5844572 was significantly higher in the p16INK4a methylated groupcompared to unmethylated group (p = 0.036), but nosignificant differences were observed when comparing
the p14ARF methylated with unmethylated groups.
Association between MIF polymorphisms and methylation
status of p14ARF or p16INK4a

By a logistic regression analysis after adjusting forconfounding factors including age, gender, clinical type,and extent of disease, carrying C allele of rs755622 wassignificantly associated with CIHM of p14ARF (Table 3;
OR, 2.16; 95% CI, 1.08–4.32; p = 0.030). By contrast, nosignificant relationship was found between p16INK4amethylation and the allele frequencies associated with
rs755622. The rs755622 CC homozygous was not(2020) 21:201

Sakurai et al. BMC Medical Genetics

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Table 2 Demographic characteristics, allelic frequencies, and CDKN2A methylation status
Overall UC

p14-UM

p14-M

Number of sample

159

105

54

Mean age ± SD

41.3 ± 13.6

40.4 ± 13.4

43.0 ± 14.0

Male: female

91: 68

66: 3925: 29Clinical typepa

p16-UM
89

70

NS

42.1 ± 15.2

40.2 ± 11.3

NSNS

50: 3941: 29

NS

p16-M

NS

NS

Chronic continuous

56

38

18

31

25

Relapse/remitting

103

67

36

58

45

Total colitis

74

49

25

35

39

Distal or left side colitis (rs755622 G > C)

85

56

29

GG

93

68

25

Disease extension

NS

NS

54

31

0.010

57

36

28

29

4

5

NS
NS

GC

57

32

25

CC

9

5

4

NS
0.050

C allele freqency (rs5844572 CATT repeat)

23.6%

20.0%

30.6%

20.2%

27.9%

5/5

16

13

3

9

7

5/6

57

40

17

31

26

5/7

28

16

12

12

16

6/6

28

19

9

23

5

6/7

23

14

9

12

11

7/7

7

3

4

2

5

5 repeat freqency

36.8%

39.0%

32.4%

34.3%

40.0%

6 repeat freqency

42.8%

43.8%

40.7%

7 repeat freqency

20.4%

17.1%

26.9%

ARF

ARF

pb

NS

50.0%

42.1%

15.7%

26.4%

NS

0.036

INK4a

p14-UM, p14 unmethylated; p14-M, p14 methylated; p16-UM, p16
unmethylated;
p16-M, p16INK4a methylated; ap14-UM vs. p14-M; bp16-UM vs. p16-M; NS Not significant

associated with CIHM of both p14ARF and p16INK4aby arecessive genetic model.
We previously revealed that CATT 7-repeat allele of
rs5844572 promotes inflammation. Thus, we assessed theinfluence of the 7-repeat allele. Our findings indicate thatcarrying the rs5844572 7-repeat allele was a significant risk
factor for p16INK4a methylation by an adjusted logistic regression analysis (Table 4; OR, 2.57; 95% CI, 1.26–5.24; p =0.0094). By contrast, there were no significant relationships
between p14ARF methylation and rs5844572 allele frequencies. The homozygous of rs5844572 7-repeat allele was not
associated with CIHM of both p14ARF and p16INK4a.

Demographic characteristics and allele frequencies ofsubjects demonstrating no methylation or methylation ofboth p14ARF and p16INK4a

Comparisons among groups demonstrating methylation of both p14ARF and p16INK4a with those inwhich both were unmethylated are shown in Table 5.
The allele distribution of MIF (rs755622) in bothmethylated and neither methylated groups met thecriteria for HWE (p = 0.73 and p = 0.72, respectively).
There were no significant differences with respect toclinicopathological backgrounds between these twogroups.

Table 3 Association between MIF rs755622 and CDKN2A methylation
Genotype

GG vs. GC + CC

GG + GC vs. CC

GG

GC

CC

adjusted OR* (95%CI); p value

adjusted OR* (95% CI); p value

p14-UM (105)

68

32

5

referencereference

p14-M (54)

25

25

4

2.16 (1.08–4.32); p = 0.030

2.30 (0.52–10.3); p = 0.27

p16-UM (89)

57

28

4

referencereference

p16-M (70)

36

29

5

1.90 (0.974–3.69); p = 0.060

2.25 (0.52–9.69); p = 0.28

*by logistic regression analysis after adjustment for age, gender, clinical type and disease extension
UM Unmethylated, M Methylated

(2020) 21:201

Sakurai et al. BMC Medical Genetics

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Table 4 Association between MIF rs5844572 and CDKN2A methylation
Genotype (repeat number)

X/X vs. X/7 + 7/7

X/X + X/7 vs. 7/7

5/5

5/6

5/7

6/6

6/7

7/7

adjusted OR* (95% CI); p value

adjusted OR* (95% CI); p value

p14-UM (105)

13

40

16

19

14

3

referencereference

p14-M (54)

3

17

12

9

9

4

1.77 (0.864–3.63); p = 0.12

4.51 (0.79–25.7); p = 0.090

p16-UM (89)

9

31

12

23

12

2

referencereference

p16-M (70)

7

26

16

5

11

5

2.57 (1.26–5.24); p = 0.0094

5.51 (0.90–33.9); p = 0.066*by logistic regression analysis after adjustment for age, gender, clinical type and disease extension
UM Unmethylated, M Methylated, X: 5 or 6 repeat allele; 7: 7 repeat allele

The minor allele frequencies associated with
rs755622 were significantly higher in the group inwhich both p14ARF and p16INK4a were methylatedcompared to the fully unmethylated group (p = 0.029);
the C allele carrier was detected at significantly
higher frequency (p = 0.020). Similarly, the frequencyof the rs5844572 7-repeat allele was significantly
higher in the group in which both p14ARF and
p16INK4a were methylated compared to the fully
unmethylated group (p = 0.0090).

Association between MIF polymorphisms and CDKN2A
methylation

The results of an analysis in which confounding factors
were adjusted revealed that carrying the rs755622 C allele and the rs5844572 7-repeat allele was significantly
associated with an increased methylation of both p14ARF
and p16INK4a (OR, 2.70; 95% CI, 1.22–6.01; p = 0.015 andOR, 2.87; 95% CI, 1.25–6.62; p = 0.013, respectively;
Table 6). In addition, homozygous of rs5844572 7-repeat
allele was significantly associated with CIHM of bothgenes (OR, 12.0; 95%CI, 1.55–92.2; p = 0.017).

Table 5 Demographic characteristics and allele frequencies of subjects demonstrating no methylation or methylation of both p14ARF
and p16INK4a
Neither methylated

Both methylated

Number of sample

77

42

Mean age ± SD

41.6 ± 14.9

42.5 ± 12.9

Male: female

46: 31

21: 21

Clinical type25

12

Relapse/remitting

52

30

31

21

Extension

Distal or left side colitis (rs755622 G > C)

NS
NS
NS

Chronic continuous

Total colitis

p value

NS

46

21

GG

49

17

GC

24

21

CC

4

4

NS

C allele freqency (rs5844572 CATT repeat)

20.8%

34.5%

0.029

5/5

7

1

5/6

27

13

5/7

12

12

6/6

19

5

6/7

10

7

7/7

2

4

5 repeat freqency

34.4%

32.1%

6 repeat freqency

48.7%

35.7%

7 repeat freqency

16.9%

32.1%

p value: unmethylated vs. both methylated

0.020

0.0090Sakurai et al. BMC Medical Genetics(2020) 21:201

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Table 6 Association between MIF polymorphisms and CDKN2A methylation
rs755622

GG vs. GC + CC

GG + GC vs. CC

adjusted OR* (95% CI); p value

adjusted OR* (95% CI); p value

Neither methylated (77)referencereference

Both methylated (42)2.70 (1.22–6.01); p = 0.015

3.92 (0.76–20.3); p = 0.10

rs5844572

X/X vs. X/7 + 7/7

X/X + X/7 vs. 7/7

adjusted OR* (95% C.I.); p value

adjusted OR* (95% C.I.); p value

Neither methylated (77)referencereference

Both methylated (42)2.87 (1.25–6.62); p = 0.013

12.0 (1.55–92.2); p = 0.017*by logistic regression analysis after adjustment for age, gender, clinical type and disease extension
X: 5 or 6 repeat allele; 7: 7 repeat allele

Association between MIF polymorphisms and CDKN2A
methylation in phenotype of UC

Next, we investigated in what kind of UC phenotype thesignificant association of MIF polymorphisms with
CIHM of CDKN2A was seen (Table 7). Carrying
rs755622 C allele was significantly associated with CIHM

in chronic continuous of clinical type and total colitis(OR, 25.9; 95% CI, 2.55–262.6; p = 0.0059 and OR, 4.38;
95% CI, 1.12–17.2; p = 0.034, respectively). Meanwhile,
carrying 7-repeat allele of rs5844572 was significantly associated in chronic continuous type (OR, 14.5; 95%CI,
1.46–144.3; p = 0.022).

Table 7 Association between MIF polymorphisms and CDKN2A methylation in UC phenotype
rs755622

genotype

Chronic continuous

adjusted OR (95% C.I.); p value

GG

GC

CC

GG vs. GC + CC

Neither methylated (25)

17

6

2

reference

Both methylated (12)

2

6

4

25.9 (2.55–262.6); p = 0.0059a

GG

GC

CC

GG vs. GC + CC

Relapse/remitting
Neither methylated (52)

32

18

2

reference

Both methylated (30)

15

15

0

1.93 (0.732–5.07); p = 0.18a

Total colitis

GG

GC

CC

GG vs. GC + CC

Neither methylated (31)

21

10

0

reference

Both methylated (21)

9

12

0

4.38 (1.12–17.2); p = 0.034b

Distal or left side colitis

GG

GC

CC

GG vs. GC + CC

Neither methylated (46)

28

14

4

reference

Both methylated (21)

8

9

4

2.75 (0.899–8.43); p = 0.076b

X/7

7/7

rs5844572

genotype

Chronic continuous

X/X
15

8

2

reference

Both methylated (12)

3

5

4

14.5 (1.46–144.3); p = 0.022a

X/X

X/7

7/7

X/X vs. X/7 + 7/7

Neither methylated (52)

38

14

0

reference

Both methylated (30)

16

14

0

2.64 (0.970–7.17); p = 0.058a

X/X

X/7

7/7

X/X vs. X/7 + 7/7

Total colitis
Neither methylated (31)

21

10

0

reference

Both methylated (21)

10

11

0

3.47 (0.895–13.4); p = 0.072b

Distal or left side colitis

X/X

X/7

7/7

X/X vs. X/7 + 7/7

Neither methylated (46)

32

12

2

reference

Both methylated (21)

9

8

4

2.89 (0.931–8.98); p = 0.066b

adjusted for age, gender and disease extension
adjusted for age, gender and clinical type
X: 5 or 6 repeat allele; 7: 7 repeat alleleb

X/X vs. X/7 + 7/7

Neither methylated (25)

Relapse/remittingaadjusted OR (95% C.I.); p value

Sakurai et al. BMC Medical Genetics(2020) 21:201

Discussion
In the present study, we investigated the impact of MIF
gene polymorphisms on aberrant methylation in the promoter regions of p14ARF and p16INK4a, each generated byalternative splicing at the CDKN2A locus, in a cohort of
159 patients diagnosed with UC. Our results revealed that
MIF rs755622 C and rs5844572 7-repeat alleles were associated with p14ARF and p16INK4a methylation, respectively.
Furthermore, the rs755622 C and rs5844572 7-repeat alleles were both associated with enhanced CDKN2A
methylation among patients in which both p14ARF and
p16INK4a were methylated compared to those in which
neither of the sites were methylated using a dominant
genetic model. We suspect that no significant association
between homozygous of both genotypes and CIHM of
CDKN2A based on a recessive genetic model may be due
to a small number of subjects in this study.
MIF is a proinflammatory cytokine that promotes recruitment of neutrophils and macrophages to inflammatory foci in the setting of inflammatory disease [24].
Several studies have focused on MIF as a key molecule
promoting pathogenesis of a diverse array of diseases, including rheumatoid arthritis [25] and septic shock [26].
MIF is also a critical mediator of UC [4, 5, 27]. Renner
et al. reported that polymorphisms in the human MIF
gene were associated with susceptibility to and severityof several inflammatory diseases, including UC [6]. Likewise, Donn et al. revealed by promoter sequence analysis
that change of G to C at − 173 (rs755622) has a direct
impact on MIF expression as it creates a potential binding site for the transcription factor, AP-4; transcriptional
activity of the MIF gene increases in accordance with
the number of sequence repeats associated with thers5844572 polymorphism [28]. Similarly, Amoli et al. reported that a MIF promoter with rs5844572 5-repeat
was less transcriptionally active than those with 6- and7-repeats [29]. In GTEx portal site (https://gtexportal.
org/), an increased number of rs755622 minor allele correlates to the increased expression of MIF, although thedata of rs5844572 is not shown. Since the rs755622 C
and rs5844572 7-repeat alleles are in strong linkage disequilibrium [6], the combination of these two alleles
may constitute an inflammatory haplotype. This isconsistent with the reported associations linking thers755622 C- and rs5844572 7-repeat haplotype with susceptibility to juvenile idiopathic arthritis [28] as well as
to findings in our previous studies focused on gastric inflammation and carcinogenesis [30, 31]. However, in ourprevious study [7], genetic polymorphisms in MIF were
not strongly associated with susceptibility to UC; likewise, Nohara et al. reported no differences with respect
to the distribution of the rs755622 genotype when comparing findings from healthy subjects to those diagnosed
with UC patients from the general Japanese population

Page 7 of 9[32]. The results of these studies suggest that the proinflammatory haplotype of MIF may not be significantly
involved in susceptibility to UC in the Japanese
population.
The p14ARF and p16INK4a proteins are encoded by
CDKN2A by alternative splicing; these proteins act on the
p53 and pRb pathways, respectively, to promote negative
regulation of the cell cycle [33, 34]. As such, methylationmediated silencing of gene expression may have important
implications with respect to carcinogenesis. Poi et al. haveshown that methylation at each promoter site has resulted
in gene deletion or silencing in association with several
cancers [35]. Conversely, gene methylation has been
associated with chronic inflammation [36], and methylation of both p14ARF and p16INK4a is already enhanced in
non-neoplastic colonic mucosa of patients with UC [37,38]. Of these two loci, methylation at p16INK4a seems to
be of greater importance with respect to the developmentof CAC [39]. However, methylation at p14ARF may also
have important implications; methylation of both p14ARF
and p16INK4a was reported among the more invasive
forms of sporadic colorectal cancer [40]. Our present observations revealed a significant relationship between the
proinflammatory allele of MIF and methylation of both
p14ARF and p16INK4a in the colonic mucosa of patients diagnosed with UC. These findings stand in contrast to
those reported in our previous study [7], in which we
found that these alleles were not significantly associatedwith susceptibility to UC. Taken together, we infer fromthese results that carrying the proinflammatory allele of
MIF may be involved in the intensity of inflammation observed after the onset of UC among those in the Japanese
population; this allele may not be involved in the development of UC, but is involved in promoting CDKN2A
methylation. Although it is unclear whether methylation
at these sites is directly involved in the development of
CAC in patients with UC, it remains possible that individuals carrying an inflammatory allele in MIF may
be at higher risk for this complication. Furthermore,in our results, the significant association of MIF polymorphisms with CIHM of CDKN2A were found inchronic continuous of clinical type and total colitis
phenotype. Rogler has been reported continuous severe inflammation is involved in the development of
CAC in UC [41]. Meanwhile, it is well known thatthe extent of inflammatory colonic mucosa is related
to the increased risk for the development of CAC in
UC [12]. These facts suggest that the patients withchronic continuous phenotype and total colitis of UC
have a higher risk for development of CAC than withrelapse/remitting phenotype and left sided/ distal colitis of UC, respectively. MIF polymorphisms may contribute to further increasing the high risk for thedevelopment of CAC via CIHM of CDKN2A.

Sakurai et al. BMC Medical Genetics(2020) 21:201

In our present study, we focused on the CIHM of
CDKN2A. However, there is possibility that CpG islandsof many other genes are methylated in inflammatory
mucosa of UC. Recently, Tahara, one of the co-authors
in this study, revealed a high rate of hypermethylation in
the severe phenotype of UC, particularly at the CpG
islands, by genome-wide methylation analysis, and thatthese methylated genes were related to those involved inbiosynthetic processes, the regulation of metabolic processes, and nitrogen compound metabolic processes[42]. In addition, we have already reported that function
gain genotypes of various immune- or inflammationrelated molecules were associated with an increased
CpG methylation of CDH1, encoding e-cadherin, and
CDKN2A [20, 43]. Further studies for an association ofvarious genotypes with CpG islands methylation of the
responsible genes for development of CAC will beneeded.
There are various limitations to this study. First, thestudy a retrospective and utilized previously-stored tissue samples collected at a single institution in Japan. A
multi-centered prospective study based on these findings
should be conducted in the near future. Second, as thisstudy was conducted using a small number of samples,
we were unable to examine other gene polymorphismsthat might influence the methylation status of the MIF
gene. As above, a multi-centered study may provide
more samples for evaluation. Third, patients enrolled inthis study have taken various medications, not the same
medications. In addition, since the onset age of our patients was partially unclear, the analysis could not be
performed using disease duration as a co-variate. Finally,a full evaluation of the risk of developing CAC from UC
would include patients with CAC as well as those diagnosed with a precancerous condition. Again, due to thevery limited number of samples from patients who developed CAC at our institution, we were unable to study
this phenomenon directly. As such, we included samples
from patients with precancerous conditions as a next
best practice.

Conclusions
In conclusion, our findings indicate that the rs755622
C–rs5844572 7-repeat MIF haplotype, which includes
two distinct alleles that are in strong linkage disequilibrium, is significantly associated with increased methylation of both p14ARF and p16INK4a. These observations
suggest that UC patients with this inflammatory genotype of MIF may be at a higher risk for developing CAC.
Abbreviations
UC: Ulcerative colitis; MIF: Macrophage migration inhibitory factor;
AP: Activator protein; CAC: Colitis-associated-cancer; CIHM: CpG island hypermethylation; CDKN2A: Cyclin Dependent Kinase Inhibitor 2A;
PCR: Polymerase chain reaction; MSP: Methylation-specific RCR; PCR-

Page 8 of 9

SSCP: PCR- single-strand conformation polymorphism; HWE: Hardy–Weinberg
equilibrium; OR: Odds ratio; CI: Confidence interval; pRB: Retinoblastoma
protein.
Acknowledgments
Not Applicable.
Authors’ contributions
NS determined the genotype, analyzed the data and wrote the paper. TA
was responsible for instructing on the scientific research and writing of themanuscript. MN, TO, HT, TH, MO, TN-H, RH and TS2 contributed to the literature review; data analysis; drafting, editing, and critical revision of the manuscript; and approval of the final version of the manuscript. TT and TS1
obtained the clinical samples and data, and participated in the design of thestudy. All authors have read and approved the final manuscript.
Funding
No funding was received.
Availability of data and materials
All data generated during this study are included in this published article.
The raw data analyzed during the current study are not publicly available
due to risk of compromising individual privacy. The application and thewritten consent forms state that the data will only be available to theresearchers within the project. For inquires on the data, researchers should
first reach out to the owner of the database, Fujita Health University. Pleasereach out to the corresponding author with requests and for assistance withdata requests.
Ethics approval and consent to participate
The Ethics Committees of Fujita Health University approved the protocol(HM18–094), and all participants gave their written informed consent.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku,
Uchinada-machi, Ishikawa 920-0293, Japan. 2Department of Gastroenterology,
Fujita Health University, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake
470-1192, Japan. 3Department of Gastroenterology and Hepatology, Kansai
Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan.
Received: 11 August 2020 Accepted: 6 October 2020

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